RNAi probes targeting cancer-related proteins

ABSTRACT

RNAi sequences that are useful as therapeutics in the treatment of cancers of various types, including prostate cancer, sarcomas such as osteosarcoma, renal cell carcinoma, breast cancer, bladder cancer, lung cancer, colon cancer, ovarian cancer, anaplastic large cell lymphoma and melanoma; and Alzheimer&#39;s disease. These sequences target clusterin, IGFBP-5, IGFBP-2, both IGFBP-2 and -5 simultaneously, Mitf, and B-raf. The invention further provides for the use of these RNAi sequences in the treatment of cancers of various types, including prostate cancer, sarcomas such as osteosarcoma, renal cell carcinoma, breast cancer, bladder cancer, lung cancer, colon cancer, ovarian cancer, anaplastic large cell lymphoma and melanoma; and Alzheimer&#39;s disease, and a method of treating such conditions through the administration of the RNA molecules with RNAi activity to an individual, including a human individual in need of such treatment.

This application claims the benefit and priority of U.S. ProvisionalApplications Nos. 60/405,193, filed Aug. 21, 2002, 60/408,152 filed Sep.3, 2002, and 60/472,387, filed May 20, 2003, all of which areincorporated herein by reference.

BACKGROUND OF THE INVENTION

This application relates to short double stranded RNAi probes useful incancer therapy and treatment of other diseases. RNA interference or“RNAi” is a term initially corned by Fire and co-workers to describe theobservation that double-stranded RNA (dsRNA) can block gene expressionwhen it is introduced into worms (Fire et al. (1998) Nature 391,806-811, incorporated herein by reference). dsRNA directs gene-specific,post-transcriptional silencing in many organisms, including vertebrates,and has provided a new tool for studying gene function. RNAi involvesmRNA degradation, but many of the biochemical mechanisms underlying thisinterference are unknown. The use of RNAi has been further described inCarthew et al. (2001) Current Opinions in Cell Biology 13, 244-248, andElbashir et al. (2001) Nature 411, 494-498, both of which areincorporated herein by reference.

Within any given mRNA molecule, there are sites which are affected byRNAi probes, and sites which are not. Thus, one cannot simply chop upthe overall sequence into subsequences of appropriate lengths (forexample, 21 to 23 base pairs) to arrive at functional RNAi-basedtherapeutics. Indeed, published US Patent Application 2002-0086356-A1discloses a method for use in assessing where target sites might belocated in a mRNA sequence, although this method is not the onlyapproach to development of effective RNAi sequences.

SUMMARY OF THE INVENTION

The present invention provides RNAi sequences that are useful astherapeutics in the treatment of cancers of various types, includingprostate cancer, sarcomas such as osteosarcoma, renal cell carcinoma,breast cancer, bladder cancer, lung cancer, colon cancer, ovariancancer, anaplastic large cell lymphoma and melanoma; and Alzheimer'sdisease. These sequences target clusterin, IGFBP-5, IGFBP-2, bothIGFBP-2 and -5 simultaneously, Mitf, and B-raf.

The invention further provides for the use of these RNAi sequences inthe treatment of cancers of various types, including prostate cancer,sarcomas such as osteosarcoma, renal cell carcinoma, breast cancer,bladder cancer, lung cancer, colon cancer, ovarian cancer, anaplasticlarge cell lymphoma and melanoma; and Alzheimer's disease, and a methodof treating such conditions through the administration of the RNAmolecules with RNAi activity to an individual, including a humanindividual in need of such treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows relative growth rate, estimated by cell number counting ofSa OS, KH OS and U-2 OS cells following siRNA-mediated clusterin geneexpression silencing. 5×10³ cells/cell line were seeded in 6 well platesand after siRNA treatment for 70 hours the total number of cells wascounted. A significant reduction in the cell number, that is morepronounced in U-2 OS cells was observed in all clusterin knock-downcells.

FIGS. 1B and C show endogenous DNA synthesis levels and spontaneousapoptosis in clusterin knock down KH OS and U-2 OS cells, as estimatedby cell proliferation ELISA BrdU colorimetric immunoassay and a CellDeath detection ELISA photometric enzyme immunoassay, respectively.Clusterin knock-down was accompanied by reduced DNA synthesis and anenhanced rate of endogenous spontaneous apoptosis in both cell lines.

FIGS. 2A and B show reduced clonogenic potential of clusterin knock downOS cells. After siRNA treatment for 70 hours, 5×10³ KH OS cells wereseeded in 6 well plates, and the total number of cells was counted after5 days of growth in complete medium (FIG. 2A). FIG. 2B shows the resultsof a comparable experiment with U-2 OS cells.

FIGS. 3A-F show the effect of DXR treatment in OS cells and cellsensitization to DNA damage and oxidative stress followingsiRNA-mediated clusterin knock down. The dark bars in FIGS. 3A and Bshow the results when 2×10⁴ KH OS or U-2 OS cells were seeded in 6 wellplates in complete medium, siRNA treated for 70 hours and then allowedto recover. The cells were then exposed to 0.35 μM DXR for 24 hours,sub-cultured in complete medium for 72 hours and counted. The light barsin FIGS. 3A and B show the results when 2×10⁴ KH OS or U-2 OS cells wereseeded in 6 well plates in complete medium, siRNA treated for 70 hoursand DXR was added directly to the transfection medium to a finalconcentration of 0.35 μM. Cells were incubated in the drug containingtransfection medium for 24 hours, washed, allowed to recover in completemedium for 72 hours and counted.

FIG. 4 shows quantitative analysis of sequence-specific clusterin genesilencing by siRNA in PC3 tumor cells.

FIG. 5 shows the effects of paclitaxel treatment on clusterin knock downPC3 cell growth and apoptosis. Cells were treated with 50 nM of theCl-III, Cl-IV or scrambled control siRNA for 1 day. Two days followingthe siRNA treatment, cells were exposed to the indicated concentrationsof paclitaxel for 48 hours, and cell viability was determined by an invitro MTT assay.

FIG. 6 shows quantitative results of exposure of PC3 prostate cancercells to CLU-5 siRNA (Seq ID NOs. 9 and 10).

FIG. 7 shows quantitative results of exposure of A549 lung cancer cellsto CLU-5 siRNA (Seq ID NOs. 9 and 10).

FIG. 8 shows the reduction in clusterin transcript as a result oftreatment of PC3 cells with clusterin-targeted siRNA as determined byRT-PCR.

FIG. 9 shows the reduction in clusterin transcript as a result oftreatment of A549 cells with clusterin-targeted siRNA as determined byRT-PCR.

FIG. 10 shows the reduction in clusterin transcript as a result oftreatment of PC3 cells with clusterin-targeted siRNA as determined byNorthern blot.

FIG. 11 shows cell viability following treatment of PC3 cells withcombinations of siRNA and Taxol.

FIG. 12 shows cell viability following treatment of A549 cells withcombinations of siRNA and Taxol.

FIG. 13 shows the reduction in clusterin transcript as a result oftreatment of OVCAR3 cells with clusterin-targeted siRNA as determined byNorthern blot.

FIG. 14 shows the reduction in clusterin transcript as a result oftreatment of MDA-MB 231 cells with clusterin-targeted siRNA asdetermined by Northern blot.

FIG. 15 shows the reduction in clusterin transcript as a result oftreatment of MDA-MB 231 cells with clusterin-targeted siRNA asdetermined by RT-PCR.

FIG. 16 shows the reduction in clusterin transcript as a result oftreatment of MCF-7 cells with clusterin-targeted siRNA as determined byNorthern blot.

FIG. 17 shows the reduction in clusterin transcript as a result oftreatment of MCF-7 cells with clusterin-targeted siRNA as determined byRT-PCR.

FIG. 18 shows the reduction in the amount of clusterin protein in MCF-7cells treated with the siRNA relative to the scrambled control.

FIG. 19 shows the reduction in IGFBP-2 transcript as a result oftreatment of A549 cells with bispecific IGFBP-2 and -5-targeted siRNA asdetermined by RT-PCR.

FIG. 20 shows the reduction in IGFBP-5 transcript as a result oftreatment of PC3 cells with bispecific IGFBP-2 and -5-targeted siRNA asdetermined by RT-PCR.

FIG. 21 shows reduction in IGFBP-5 mRNA in PC3 cells.

FIG. 22 shows inhibitions of IGFBP-5 transcript in primary human bonefibroblast.

FIG. 23 shows growth inhibition of C42 cells by IGFBP-2/IGFBP-5bispecific siRNA.

FIG. 24 shows growth inhibition of A549 cells by IGFBP-2/IGFBP-5bispecific siRNA.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to isolated RNA molecules which mediateRNAi. That is, the isolated RNA molecules of the present inventionmediate degradation of mRNA that is the transcriptional product of thegene, which is also referred to as a target gene. For convenience, suchmRNA may also be referred to herein as mRNA to be degraded. The termsRNA, RNA molecule(s), RNA segment(s) and RNA fragment(s) may be usedinterchangeably to refer to RNA that mediates RNA interference. Theseterms include double-stranded RNA, single-stranded RNA, isolated RNA(partially purified RNA, essentially pure RNA, synthetic RNA,recombinantly produced RNA), as well as altered RNA that differs fromnaturally occurring RNA by the addition, deletion, substitution and/oralteration of one or more nucleotides. Such alterations can includeaddition of non-nucleotide material, such as to the end(s) of the RNA orinternally (at one or more nucleotides of the RNA). Nucleotides in theRNA molecules of the present invention can also comprise non-standardnucleotides, including non-naturally occurring nucleotides ordeoxyribonucleotides. Collectively, all such altered RNAi compounds arereferred to as analogs or analogs of naturally-occurring RNA. RNA of thepresent invention need only be sufficiently similar to natural RNA thatit has the ability to mediate RNAi. As used herein the phrase “mediateRNAi” refers to and indicates the ability to distinguish which mRNA areto be affected by the RNAi machinery or process. RNA that mediates RNAiinteracts with the RNAi machinery such that it directs the machinery todegrade particular mRNAs or to otherwise reduce the expression of thetarget protein. In one embodiment, the present invention relates to RNAmolecules that direct cleavage of specific mRNA to which their sequencecorresponds. It is not necessary that there be perfect correspondence ofthe sequences, but the correspondence must be sufficient to enable theRNA to direct RNAi inhibition by cleavage or lack of expression of thetarget mRNA.

As noted above, the RNA molecules of the present invention in generalcomprise an RNA portion and some additional portion, for example adeoxyribonucleotide portion. The total number of nucleotides in the RNAmolecule is suitably less than 49 in order to be effective mediators ofRNAi. In preferred RNA molecules, the number of nucleotides is 16 to 29,more preferably 18 to 23, and most preferably 21-23.

A first group of RNA molecules in accordance with the present inventionare directed to mRNA encoding clusterin, a protein also known astestosterone-repressed prostate message-2 (TRRM-2) or sulfatedglycoprotein-2 (SGP-2). Clusterin is expressed in increased amounts byprostate tumor cells following androgen withdrawal. Furthermore, it hasbeen determined that antisense therapy which reduces the expression ofclusterin provides therapeutic benefits in the treatment of cancer. Inparticular, such antisense therapy can be applied in treatment ofprostate cancer and renal cell cancer. (PCT Patent Publication WO00/49937, which is incorporated herein by reference). Administration oftherapeutic agents clusterin also can enhance sensitivity of cancercells to chemotherapeutic agents and to radiotherapy both in vitro andin vivo. Sequences of specific RNA molecules which may be used tointerfere with the expression of clusterin are listed in Table 1 and 7.(See, U.S. patent application Ser. No. 09/967,726 which is incorporatedherein by reference) These sequences can be used alone or in combinationwith other chemotherapy agents or apoptosis inducing treatment conceptsin the treatment of prostate cancer, sarcomas such as osteosarcoma,renal cell carcinoma, breast cancer, bladder cancer, lung cancer, coloncancer, ovarian cancer, anaplastic large cell lymphoma and melanoma. Inaddition, clusterin has been shown to promote amyloid plaque formationand to be critical for neuritic toxicity in mouse models for Alzheimer'sdisease. (De Mattos et al., Proc. Nat'l Acad. Sci. (USA) 99: 10843-10848(2002), which is incorporated herein by reference). Thus, the sequencesof the invention can also be used in the treatment of Alzheimer'sdisease.

TABLE 1 Clusterin RNAi Sequences Target region 487-505 1105-11231620-1638 of cDNA (nt) sense siRNA ccagagcucgcccuucuac-dtdtgaugcucaacaccuccucc-dtdt cuaauucaauaaaacuguc-dtdt (SEQ. ID No: 1) (SEQID No: 3) (SEQ. ID No: 5) antisense guagaagggcgagcucugg-dtdtggaggagguguugagcauc-dtdt gacaguuuuauugaauuag-dtdt siRNA (SEQ. ID No: 2)(SEQ. ID No: 4) (SEQ. ID No: 6) Target region HIV 1152-1176   53-71 notsequence specific of cDNA (nt) sense siRNA uaauucaacaaaacugu-dtdtaugaugaagacucugcugc-dtdt ugaaugaagggacuaaccug-dtdt (SEQ. ID No: 7) (SEQ.ID No: 9) (SEQ. ID No: 11) antisense acaguuuuguugaauua-dtdtgcagcagagucuucaucau-dtdt cagguuagucccuucauuca-dtdt siRNA (SEQ. ID No: 8)(SEQ ID No: 10) (SEQ. ID No: 12) Target region not sequence specific notsequence specific of cDNA (nt) sense siRNA cagaaauagacaaagugggg-dtdtacagagacuaagggaccaga-dtdt (SEQ. ID No: 13) (SEQ. ID No: 15) antisenseccccacuuugucuauuucug-dtdt acagagacuaagggaccaga-dtdt siRNA (SEQ. ID No:14) (SEQ. ID No: 16)

Specific results relating to the use of the RNAi species shown in Table7 are shown in the Figures. These results demonstrate the effectivenessof clusterin suppression by RNAi mediated processes to reduce the growthof and to promote apoptosis in osteosarcoma cells, thus demonstrating afurther type of condition which can be treated in accordance with theinvention. The results also demonstrate the RNAi treatment to reduceclusterin levels results in increased levels of p53 and reduced levelsof bcl-2. Thus, in accordance with the invention, conditions in whichactive p53 or bcl-2 levels are affected due to regulation as opposed toa mutation that renders the tumor suppressor wholly or partiallyinactive are overcome by administration of an amount of clusterin RNAieffective to reduce the amount of clusterin present, and thus theundesirable clusterin-associated modulation of p53 and bcl-2 levels.

A second group of RNA molecules in accordance with the present inventionare directed to mRNA encoding insulin-like growth factor bindingprotein-5 (IGFBP-5). It has been shown that inhibition of IGFBP-5expression can delay the progression of hormone-regulated (prostatic orbreast) tumor cells to hormone (e.g. androgen or estrogen) independence,provide a therapeutic method for the treatment of individuals, includinghumans, suffering from hormone regulated cancers, such as breast orprostate cancer and inhibit or delay the growth and metastaticprogression of prostate, breast and other IGF-1 sensitive tumors inbone. (Published PCT Application No. WO01/05435, which is incorporatedherein by reference.) These same results are obtained using RNAi therapyin accordance with the invention using siRNA molecules having thesequences set forth in Table 2. These sequences can be used alone or incombination with other chemotherapy agents or apoptosis inducingtreatment concepts.

TABLE 2 IGBFP-5 RNAi Sequences Target region   44-61 876-895 notsequence specific of cDNA (nt) sense siRNA augguguugcucaccgcg-dtdtcccugggcugcgagcugguc-dtdt gaggaaacugaggaccucgg-dtdt (SEQ. ID No: 17)(SEQ. ID No: 19) (SEQ. ID No: 21) antisense cgcggugagcaacaccau-dtdtgaccagcucgcagcccaggg-dtdt ccgagguccucaguuuccuc-dtdt siRNA (SEQ. ID No:18) (SEQ. ID No: 20) (SEQ. ID No: 22) Target region not sequencespecific 850-568 1225-1243 of cDNA (nt) sense siRNAcucggauucucaugcaaggg-dtdt agcccucuccaugugcccc-dtdtgaagcugacccaguccaag-dtdt (SEQ. ID No: 23) (SEQ. ID No: 25) (SEQ. ID No:27) antisense cccuugcuagagauuccgag-dtdt ggggcacauggagagggcu-dtdtcuuggacugggucagcuuc-dtdt siRNA (SEQ. ID No: 24) (SEQ. ID No: 26) (SEQ IDNo: 28) Target region 1501-1520 of cDNA (nt) sense siRNAgcugccaggcauggaguacg-dtdt (SEQ. ID No: 29) antisensecguacuccaugccuggcagc-dtdt siRNA (SEQ. ID No: 30)

A third group of RNA molecules in accordance with the present inventionare directed to mRNA encoding insulin-like growth factor bindingprotein-2 (IGFBP-2). It has been shown that inhibition of expression ofIGFBP-2 delays the progression of prostatic tumor cells to androgenindependence, and provides a therapeutic benefit for mammalianindividuals, including humans, suffering from hormone-regulated cancersuch as prostate or breast cancer. In addition, the compositions of theinvention can be used to inhibit or delay the growth and metastaticprogression of such cancers, (Published PCT Application No. WO02/22642,which is incorporated herein by reference). These same results areobtained using RNAi therapy in accordance with the invention using siRNAmolecules having the sequences set forth in Table 3. These sequences canbe used alone or in combination with other chemotherapy agents orapoptosis inducing treatment concepts.

TABLE 3 IGFBP-2 RNAi Sequences Target region 118-138 1393-1411 906-924of cDNA (nt) sense siRNA augcugccgagagugggcugcd-TdTccccugugucccuuuugca-dTdT cugugacaagcauggccug-dTdT (SEQ. ID No: 31) (SEQ.ID No: 33) (SEQ. ID No: 35) antisense gcagcccacucucggcagcau-dTdTugcaaaagggacacagggg-dTdT caggccaugcuugucacag-dTdT siRNA (SEQ. ID No: 32)(SEQ. ID No: 34) (SEQ. ID No: 36) Target region 525-542 of cDNA (nt)sense siRNA gcgccgggacgccgagua-dTdT (SEQ. ID No: 37) antisenseuacucggcgucccggcgc-dTdT siRNA (SEQ. ID No: 38)

A fourth group of RNA molecules in accordance with the present inventionare directed to mRNA encoding insulin-like growth factor-2 and 5simultaneously (IGF-Bis). Inhibition of expression of both IGFBP-2 andIGFBP-5 can delay the progression of hormone-regulated (prostatic orbreast) tumor cells to hormone (e.g. androgen or estrogen) independence,provide a therapeutic method for the treatment of individuals, includinghumans, suffering from hormone regulated cancers, such as breast orprostate cancer and inhibit or delay the growth and metastaticprogression of prostate, breast and other IGF-1 sensitive tumors in bonepotentially more effectively than the inhibition of either of thesefactors (Published PCT Application No. WO01/05435, Published PCTApplication No. WO02/22642, and U.S. Provisional Application No.60/350,046, filed Jan. 17, 2002, which are incorporated herein byreference.) These same results are obtained using RNAi therapy inaccordance with the invention using siRNA molecules having the sequencesset forth in Table 4. These sequences can be used alone or incombination with other chemotherapy agents or apoptosis inducingtreatment concepts.

TABLE 4 IGFBP-2 and IGFBP-5 Bispecific RNAi Sequences Target regionBP5-898-919 BP5 948-964 BP5 976-991 of cDNA (nt) BP2 346-362 BP2 416-432BP2 444-459 sense siRNA ggagccgggcugcggcugc-dtdt cgugcggcgucuacacc-dtdtccaggggcugcgcugc-dtdt (SEQ. ID No: 39) (SEQ. ID No: 41) (SEQ. ID No: 43)antisense gcagccgcagcccggcucc-dtdt gguguagacgccgcacg-dtdtgcagcgcagccccugg-dtdt siRNA (SEQ. ID No: 40) (SEQ. ID No: 42) (SEQ. IDNo: 44)

A fifth group of RNA or DNA antisense molecules in accordance with thepresent invention are directed to mRNA encoding the group ofmicrophthalmia transcription factors (Mitf). Bcl-2 is regulated inmelanoma and other cells by the master regulator Mitf which has beenreported to modulate melanoma cell viability, lineage survival, andsusceptibility to apoptosis (McGill et al. (2002) Cell 109, 707-718,incorporated herein by reference). Mitf and Bcl-2 regulated by Mitf areexpressed in increased amounts by various human tumors. RNAi orantisense therapy which reduces the expression of Mitf may providetherapeutic benefits in the treatment of cancer. In accordance with theinvention, Mitf can also enhance sensitivity of cancer cells tochemotherapeutic agents and to radiotherapy both in vitro and in vivo.Sequences of specific antisense or RNA molecules which may be used tointerfere with the expression of Mitf are listed in Table 5. Thesesequences can be used alone or in combination with other chemotherapyagents or apoptosis inducing treatment concepts in the treatment ofmelanoma, prostate cancer, renal cell carcinoma, bladder cancer, lungcancer, bone cancer and other tumors.

TABLE 5 Mitf RNAi Sequences Target region 207-225 1287-1305 2172-2190 ofcDNA (nt) sense siRNA ccgcugaagagcagcaguu-dtdt augcaggcucgagcucaug-dtdtagauacaguaccccucuag-dtdt (SEQ. ID No: 45) (SEQ. ID No: 47) (SEQ. ID No:49) antisense aacugcugcucuucagcgg-dtdt caugagcucgagccugcau-dtdtcuagagggguacuguaucu-dtdt siRNA (SEQ. ID No: 46) (SEQ. ID No: 48) (SEQ.ID No: 50)

A sixth group of RNA or DNA antisense molecules in accordance with thepresent invention are directed to mRNA encoding B-raf. B-raf is a keyplayer in cellular signal transduction and is activated by somaticmissense mutations in 66% of malignant melanomas and at lowerfrequencies in a wide range of human cancers (Davies et al. (2002)Nature 417, 949-954, incorporated herein by reference). RNAi orantisense therapy which reduces the expression of activated and/ornon-activated B-raf may provide therapeutic benefits in the treatment ofcancer. In accordance with the invention, reduction in expression ofB-raf can also enhance sensitivity of cancer cells to chemotherapeuticagents and to radiotherapy both in vitro and in vivo. Sequences ofspecific antisense or RNA molecules which may be used to interfere withthe expression of B-raf are listed in Table 6. These sequences can beused alone or in combination with other chemotherapy agents or apoptosisinducing treatment concepts in the treatment of melanoma, prostatecancer, renal cell carcinoma, bladder cancer, lung cancer, bone cancerand other tumors.

TABLE 6 b-raf RNAi Sequences Target region 362-380 1184-1202 2249-2267of cDNA (nt) sense siRNA ucucugggguucgguucug-dtdtccugucaauauugaugacu-dtdt cccuccuuguuucgggcug-dtdt (SEQ. ID No: 51) (SEQ.ID No: 53) (SEQ. ID No: 55) antisense caguuccguuccccagaga-dtdtagucaucaauauugacagg-dtdt cagcccgauucaaggaggg-dtdt siRNA (SEQ. ID No: 52)(SEQ. ID No: 54) (SEQ. ID No: 56)

The siRNA molecules of the invention are used in therapy to treatpatients, including human patients, that have cancers or other diseasesof a type where a therapeutic benefit is obtained by the inhibition ofexpression of the targeted protein. siRNA molecules of the invention areadministered to patients by one or more daily injections (intravenous,subcutaneous or intrathecal) or by continuous intravenous or intrathecaladministration for one or more treatment cycles to reach plasma andtissue concentrations suitable for the regulation of the targeted mRNAand protein.

Example 1 Protocol for Transfection of LNCaP and PC3 Cells with siRNADuplexes

1) Cell preparation:

In each well of 6-well plate seed 0.5×10⁶ of LNCaP cells [PC3 cell atthe density 0.3×10⁶ per well] in appropriate media containing 5% FBSwithout antibiotics [penicillin/streptomycin]

Incubate the cells at 37° C. in a humidified 5% CO2 incubator until theyreach 40-50% confluence.

2) si RNA preparation:

Prepare the following si RNA dilution in microcentrifuge tubes. For eachwell: 0.01-100 nM

3) Prepare the following transfection reagent dilution inmicrocentrifuge tubes:

For each well of 6-well plate dilute 4 ml of OligoFECTAMINE™ Reagentinto 11 ml of OPTI-MEM™ and incubate 10 min at room temperature.

4) Combine the diluted OligoFECTAMINE™ to the diluted siRNA duplexes andmix gently by inversion.

5) Incubate 20 min at room temperature.

6) Remove the media from the well and replace it with 800 ml ofOpti-MEM™.

7) Overlay the 200 ml of transfection complexes onto the cells.

8) Incubate 4 hrs at 37 degrees C. in a CO₂ incubator.

9) add 500 ml of media containing 15% FBS

10) after 24 hrs check gene expression by Real Time PCR or

11) check protein expression with Western Blot after 1, 6, 12, 24, 48,72 and 96 hours

Example 2

The human clusterin cDNA was manually scanned in order to identifysequences of the AA(N₁₉)UU (N, any nucleotide) type that fulfill therequired criteria for siRNA (Harbourth et al., J Cell Sci 114: 4557-4560(2001)). Two such sequences with symmetric 2-nt 3′ overhangs, were found433 (Cl-I) and 644 (Cl-II) nts downstream of the CLU gene transcriptioninitiation codon. Two additional oligonucleotides used targeted a region1620 nts downstream of the CLU gene transcription initiation codon(Cl-III) and the human CLU transcription initiation site (Cl-V) (seeTable). BLAST analysis showed no homology with other known human genes.Selected RNA oligos were synthesized by Dharmacon Research, Inc.(Lafayette, Colo.), diluted at a 20 μM final concentration in RNasefreeddH₂O and stored at −20° C. The Scramble-I™ (Sc-I) (D-1200-20) andScramble-II™ (Sc-II) (D-1205-20) oligonucleotides used were purchasedfrom Dharmacon Research, Inc.

siRNA transfection of the Cl-I, Cl-II and scrambled RNA duplexes inexponentiary growing OS cells was performed as described (Harbourth etal., Tuschl et al, Genes Dev. 13: 3191-3197 (1999)). Briefly, cells wereseeded the day before siRNA transfection in 24-well plates containing500 μl complete medium and were ˜40-50% confluent during transfection.For the transfection mixture 100 nM of siRNA duplex per well were used.The RNA duplex was diluted in Opti-MEM® I (Gibco Life Technologies,Inc., San Diego, Calif.) serum free medium and transfection efficiencywas enhanced by using the Oligofectamine™ reagent (Invitrogen Lifetechnologies Inc., San Diego, Calif.). When cells were treated with bothCl-i and Cl-II oligonucleotides, 100 nM of each siRNA duplex were used.Cell treatment with the siRNA oligonucleotides lasted for 2-3 days.

Alternatively, in PC3 cells Lipofectin™ (Invitrogen Life technologiesInc., San Diego, Calif.) was used to enhance transfection with theCl-III and CL-V oligonucleotides. PC3 cells were treated with 10, 50, or100 nM of the RNA duplexes after pre-incubation with 4 μg/ml ofOligofectamine™ reagent in serum free OptiMEM® I for 20 minutes. Fourhours after starting the incubation, the medium containing the RNAduplexes and Lipofectin was replaced with standard tissue culture mediumCells were treated once on day 1 and then harvested 48 h after treatmenton day 3. In all cases controls used included: (a) the usage of the Sc-Iand Sc-II RNA duplexes and (b) mock transfections in the absence of anucleic acid (Con-I). CLU gene silencing was assayed by RNA blotanalysis, immunoblotting analysis or confocal immunofluorescence.

Efficient silencing of the CLU gene expression in OS cells is achievedusing siRNA. Treatment of the three OS cell lines with the Cl-I or theCl-II siRNA oligonucleotides appeared to be quite effective and resultedin knocking down significantly the cellular CLU protein levels.Interestingly, the Cl-I oligonucleotide appeared to be slightly moreeffective than Cl-II in silencing the CLU gene. No CLU gene silencingwas seen in the presence of the control Sc-I or the Sc-IIoligonucleotides or at the absence of RNA duplexes from the transfectionmedium.

Next we addressed the issue of whether the CLU-specific siRNAoligonucleotides could also inhibit the accumulation of the CLU proteinfollowing cellular exposure to DXR. Exposure of the KH OS and U-2 OScells to DXR for 24 h in the presence of the Cl-I, Cl-II or a mixture ofboth the Cl-I, Cl-II oligonucleotides effectively abolished CLU proteinaccumulation. It is thus evident that in the presence of the Cl-I or theCl-II oligonucleotides the cellular CLU protein cannot be induced aftercell exposure to apoptosis inducing agents.

Example 3

Phenotypic effects in OS cells following CLU gene expression silencingby siRNA. The effects of CLU gene expression silencing in OS cells werestudied by direct counting of the cells following siRNA, by recordingcellular morphology and phenotype, as well as by clonogenic assays. CLUknock down in KH OS and Sa OS cells did not result in any visiblephenotype. However, the CLU knock down cells were found to besignificantly growth retarded as compared to their control counterparts(FIG. 1A). In contrast at the U-2 OS cells, that express the higherendogenous amount of the CLU protein, the effects of CLU knock downappeared quite significant. Specifically, CLU siRNA treated (for threedays) U-2 OS cells lost their firm adherence to plastic and acquired arounding shape. This phenotype was accompanied by a severe growthretardation effect. In order to study whether a combination of both Cl-Iand Cl-II RNA duplexes would be more effective in inhibiting cellgrowth, we treated cells with both these oligonucleotides. For the KH OSand U-2 OS cells, only a slight increase in growth retardation wasobserved as compared to the Cl-I treated cells. Finally, in order todistinguish between the cytostatic and cytotoxic effects of CLU proteinelimination we directly assayed CLU knock down KH OS and U-2 OS cellsfor DNA synthesis and endogenous spontaneous apoptosis. CLU knock downcells showed a reduced DNA synthesis rate (FIG. 1A) and higher levels ofendogenous spontaneous apoptosis (FIG. 1B) as compared to their siblingcontrols. Effects were again more pronounced in U-2 OS cells. Insummary, these results suggest that the reduced number of CLU knock downcells is due to a reduced rate of cell proliferation as well as to anincreased level of spontaneous apoptosis.

The effect of CLU knock down in plating efficiency and growth followingsiRNA was also studied by clonogenic assays as recent studies havedemonstrated that gene silencing is sustained for more than 7 cellulardoublings (Harbourth et al.). KH OS and U-2 OS cells were selected forthese assays since they represent two extreme opposite cases as far asthe endogenous CLU amount and the intensity of CLU accumulation duringstress are concerned. CLU knock-down KH OS cells when plated were firmlyattached to the plastic (more than 90% of the seeded cells wereattached) and only a few of the attached cells showed an abnormalmorphology. However, the growth potential of the adherent cells wasimpaired as found 5 days post-plating after analyzing the total colonynumber and size of the formed colonies (FIG. 2A). CLU knocked-down U-2OS cells were poorly attached to the plastic after trypsinization (only−70% of the seeded cells were attached) and most of the adherent cellsappeared quite abnormal in shape. Cells showed an extremely lowproliferation potential (FIG. 2B) and after 9 days in culture only somesmall colonies could be seen.

Example 4

Sustained silencing of CLU gene expression in OS cells by siRNA resultsin significant sensitization to apoptosis induced by genotoxic andoxidative stress. Prior to CLU functional assays, we analyzed the DXReffects in OS cells since the drug-related reported effects vary indifferent cell-types (Gewritz et al, Biochem Pharmacol. 57: 727-741(1999)). As the DXR plasma concentration in treated patients fall into arange of 1-2 μM and decline into a range of 0.025-0.25 μM within 1 h(Muller et al, Cancer Chemother. Pharmacol., 32: 379-384 (1993)) cellswere treated with 0.35 and 1 μM of DXR. To analyze the extent of theDXR-mediated cell death we scored apoptosis by TUNEL. Attached cellsfollowing drug treatment for 24 h underwent significant morphologicalchanges as compared to non-treated control cells. At this time cellsexhibit an enlarged and flattened morphology that is reminiscent of asenescence-like phenotype, while a significant number of them are TUNELpositive. On-going apoptosis is also apparent 24 h later, verifying thatapoptosis is a dynamic process that continues even after DXR removalfrom the medium. In agreement with the results obtained by TUNEL, DXRtreatment was accompanied by PARP cleavage; the anti-apoptotic proteinbcl-2 showed no altered expression in drug-treated KH OS and U-2 OScells. Following DXR treatment, accumulation of p53 protein and itsdownstream effectors related to either growth arrest (p21) or apoptosis(bax) was found only in U-2 OS cells indicating that the cytostatic andcytotoxic effects mediated by DXR in OS cell lines rely on bothp53-dependent and p53-independent mechanisms.

Next, we followed two complementary approaches to study the effect ofCLU knock down in OS cells exposed to DXR (FIGS. 3A and B). SiRNAtreated cells were either re-plated in complete medium and weresubsequently exposed to 0.35 μM DXR for 24 h, or they were exposed to0.35 μM DXR in the presence of the Cl-II or Cl-I oligonucleotides. Inboth cases viable cells were counted 3 days post-DXR treatment. CLUknocked-down KH OS cells appeared more sensitive to DXR than theircontrol counterparts (FIG. 3A-dark bars), whereas DXR treatment appearedsignificantly more effective when it was combined with the presence ofthe CLU-specific siRNA oligonucleotides in the medium (FIG. 3A—lightbars). Similarly, CLU knocked-down Sa OS cells were more sensitive tothe DXR treatment. In U-2 OS cells both strategies appeared veryeffective and CLU knock-down cells were significantly more sensitive tothe drug as compared to controls (FIG. 3B). When DXR treatment wasperformed in the presence of the CLU-specific siRNA oligonucleotides,the CLU knock-down cells appeared to be significantly more sensitive tothe drug (FIG. 3B—light bars) and a massive apoptosis was observed.Finally, when U-2 OS cells were treated with both the Cl-I and Cl-IIoligonucleotides, cells were almost eliminated.

To understand the mechanism of cell sensitization following CLU knockdown, we directly assayed the intensity of apoptosis induction rightafter cell exposure to agents inducing genotoxic (DXR) or oxidativestress (H₂O₂). As shown in FIG. 3C-F. Exposure of the CLU knock-downcells to either DXR or H₂O₂ resulted in a significantly higher rate ofapoptosis in both KH OS and U-2 OS cells. This observation suggests thatCLU directly affects or interacts with the cellular machinery involvedin apoptosis, by providing cytoprotective signals.

Example 5

OS cells sensitization to genotoxic and oxidative stress due to CLU genesilencing is related to activation of the cellular apoptotic machinery.By analyzing the expression levels of other recently identified CLUprotein forms, to our surprise, we found that the Cl-I and Cl-IIoligonucleotides did not exert any significant effect on the putative 55kDa n-CLU³⁵ CLU protein form in both KH OS and U-2 OS cells; a minoreffect on the 49 kDa c-CLU⁴⁹ protein form level was detected despite thefact that the binding sites of the Cl-I or Cl-II oligonucleotides arecommon between the s-CLU and n-CLU mRNAs (Leskov et al., J. Biol. Chem278: 11590-16000 (2003)). We assume that the explanation of this effectrelies on our observation that the n-CLU protein is extremely stable.Thus CL-I and CL-II oligonucleotides specifically knock-down thesecreted CLU (s-CLU) protein form.

We then assayed the expression levels of several proteins involved inregulating apoptosis in human cells. CLU knock down in both KH OS andU-2 OS cells resulted in the down-regulation of the anti-apoptoticmolecule bcl-2. No effect was detected on levels of Ku70, a proteinimplicated in DNA damage repair and signaling that, moreover, bindsn-CLU (Yang et al., Proc. Nat'l Acad. Sci (USA) 97: 5907-5912 (2000)).Interestingly, in U-2 OS cells, which bear a functional p53 molecule,CLU knock down apart from bcl-2 down-regulation is also accompanied byp53 accumulation and up-regulation of its downstream pro-apopticeffector, bax. Supportively, CLU knock-down U-2 OS cells when exposed toDXR showed a more intense and robust accumulation of the p53 protein ascompared to the Sc-I treated cells. We suggest that sensitization of OScells following CLU knock down largely depends on the activation of thecellular pro-apoptotic machinery. On-going studies in our laboratoriesare investigating the implication of CLU on the cell signaling cascadesrelated to apoptosis regulation.

Example 6

Effects of CLU gene silencing in PC-3 prostate cancer cells weredetermined. Having established the significant effects of CLU knock downin OS cells, we then applied CLU siRNA in PC3 human prostate cancercells. PC3 cells are p53 null (Rohlff, et al., Prostate 37: 51-59(1998)) and express relatively low endogenous amount of the s-CLUprotein form similar to the Sa OS cells. In PC3 cells, apart fromemploying the Cl-I and Cl-II oligonucleotides, we also tested twoadditional CLU-specific siRNA oligonucleotides (Cl-III, Cl-V). Fromthese oligos, Cl-V targeted the s-CLU transcription initiation site.Usage of the Cl-I and Cl-II oligonucleotides in PC3 cells resulted insimilar effects to those described for OS cell lines. As it can beenseen in FIG. 4 both the Cl-III and Cl-V oligonucleotides are quiteeffective in silencing CLU RNA and protein expression in asequence-dependent but dose-independent manner. More specifically,treatment of the PC3 cells, for one day, with 10, 50 and 100 nM ofeither Cl-III or Cl-V siRMN oligonucleotides severely reduced CLU mRNAlevels ranging from 60% to 98% (FIG. 4). Tins effect on mRNA levels wasalso evident at protein level. Additionally and in agreement withfindings in the U-2 OS cells, CLU knock down in PC3 cells resulted insignificant morphologic changes that resembled an on-going apoptosis.

To determine whether treatment of PC3 cells with CLU siRNAoligonucleotides could enhance the cytotoxic effects of chemotherapeuticdrugs, PC3 cells were treated first with the Cl-III, Cl-V or the Sc-IsiRNA oligonucleotides and then incubated with medium containing variousconcentrations of Paclitaxel for 2 days. An MTT assay was then performedto determine cell viability. As shown in FIG. 5, CLU siRNA treatmentsignificantly enhanced chemosensitivity of Paclitaxel in adose-dependent manner reducing the IC₅₀ (the concentration that reducescell viability by 50%) of Paclitaxel by more than 90%, whereas thescrambled siRNA had no effect.

TABLE 7 Sequence specific characteristics pf the Cl-I, Cl-II, CL-III andCl-V oligonucleotides. The Cl-I, Cl-II, Cl-III and Cl-V clusterinspecific siRNA oligonucleotides have GC/AT ratios of 57/43, 67/33, 24/76and 43/57, respectively. Cl-I Cl-II Targeted AAccagagctcgcccttctacTTAAgtcccgcatcgtccgcagcTT region (SEQ ID No: 57) (SEQ. ID No: 60) SensesiRNA ccagagcucgcccuucuacdTdT gucccgcaucguccgcagcdTdT (SEQ. ID No: 58)(SEQ. ID No: 61) Antisense guagaagggcgagcucuggdTdTgcugcggacgaugcgggacdTdT siRNA (SEQ. ID No: 59) (SEQ. ID No: 62) Cl-IIICl-V Targeted AActaattcaataaaactgtcTT GCatgatgaagactctgctgcTG region(SEQ. ID No: 63) (SEQ. ID No: 66) Sense siRNA cuaauucaauaaaacugucdTdTaugaugaagacucugcugc (SEQ. ID No: 64) (SEQ. ID No: 67) AntisensegacaguuuuauugaauuagdTdT gcagcagagucuucaucau siRNA (SEQ. ID No: 65) (SEQ.ID No: 68)

Example 7

8 species of siRNA targeting clusterin were formed as double-strandedRNA from Seq. ID NOs. 1 and 2, 3 and 4, 5 and 6, 7 and 8, 9 and 10, 11and 12, 13 and 14, and 15 and 16, and labeled as CLU1-CLU 8,respectively. PC3 cells were transfected with various doses (10, 50 and100 nM) of the 8 species of siRNA or scrambled control. Three days aftertreatment, proteins were extracted and analyzed by Western blotting forclusterin levels (MW=40 and 60 kDa). Reduction in the amount ofclusterin was observed with all eight species of siRNA, although thebest results were obtained with CLU-5 (Seq ID Nos 9 and 10).

Densitometric measurements were performed after normalization to avinculin control the blots for cells treated with CLU-5 (Seq ID Nos. 9and 10). The results are summarized in FIG. 6. “Oligo” cells weretreated with oligoFECTAMINE™ only. As can be seen, a dose dependentresponse to the siRNA was observed.

Example 8

The experiment of Example 7 was repeated using A549 lung cancer cells inplace of PC3 prostate cells, and comparable results were observed.Densitometric measurements were performed after normalization to avinculin control the blots for cells treated with CLU-5 (Seq ID Nos. 9and 10). The results are summarized in FIG. 7. “Oligo” cells weretreated with oligoFECTAMINE™ only. As can be seen, a dose dependentresponse to the siRNA was observed.

Example 9

PC3 cells were transfected with siRNA at levels of 10, 50 or 100 nM orwith 100 nM scrambled control. Two days after transfection, total RNAwas extracted and the level of clusterin transcript was quantified byReal Time PCR. The results are shown in FIG. 8. As can be seen, each ofthe 8 species of siRNA tested resulted in a reduction in the amount ofclusterin transcript.

Example 10

The experiment of Example 9 was repeated using A549 cells in place ofPC3 cells. The results are shown in FIG. 9. As can be seen, each of the8 species of siRNA tested resulted in a reduction in the amount ofclusterin transcript.

Example 11

PC3 cells were treated (1 pulse) with CLU-3 (Seq ID NOs. 5 and 6) orCLU-5 (Seq ID Nos 9 and 10) or with a scrambled control at levels of 10,50 or 100 nM, or with oligoFECTAMINE™ only. After two days total RNA wasextracted and analyzed for clusterin and GADPH by Northern blotting.Densitometric measurement was performed. FIG. 10 presents results ofthese measurements after normalization to GADPH. Reduction in the amountof clusterin transcript was observed at all doses of CLU-3 and CLU-5.

Example 12

PC3 cells were treated once with 25 nM human clusterin siRNAs (CLU-3 andCLU-5), a scrambled control, or oligoFECTAMINE™ only. After two days oftreatment, the medium was replaced with medium containing variousconcentrations of Taxol (Paclitaxel). After three days of incubation,cell viability was determined by MTT assay. FIG. 11 summarizes theresults. As can be seen, the siRNA and the Taxol worked in synergy toreduce the number of viable cells.

Example 13

The experiment of Example 12 was repeated with A549 cells in place ofPC3 cells. The results are summarized in FIG. 12

Example 14

OVCAR3 ovarian cancer cells were treated once with 1, 10 or 50 nM CLU5,scrambled control or a vehicle only control. An untreated control wasalso run. After two days, total RNA was extracted and analyzed forclusterin and GAPDH mRNA by Northern blot. Densitometric measurements ofclusterin mRNA levels after normalization to GAPDH mRNA are shown inFIG. 13. Substantial dose dependent reduction in the amount of clusterintranscript was observed.

Example 15

MDA-MB 231 human breast cancer cells were transfected with 5, 50 or 100nM CLU-5 or a scrambled control, or with oligoFECTAMINE™ vehicle alone.Two days after transfection, RNA was extracted and analyzed forclusterin and GAPDH by Northern blotting. Densitometric measurements ofclusterin mRNA levels after normalization to GAPDH mRNA are shown inFIG. 14. Substantial reduction in the amount of clusterin transcript wasobserved.

Example 16

The experiment of Example 15 was repeated, except that clusterintranscript was quantified using RT-PCR. The results are summarized inFIG. 15. Substantial reduction in the amount of clusterin transcript wasobserved.

Example 17

MCF-7 human breast cancer cells were transfected with 5, 25 or 50 nMCLU-5 or a scrambled control, or with oligoFECTAMINE™ vehicle alone. Twodays after transfection, RNA was extracted and analyzed for clusterinand GAPDH by Northern blotting. Densitometric measurements of clusterinmRNA levels after normalization to GAPDH mRNA are shown in FIG. 16.Substantial dose-dependent reduction in the amount of clusterintranscript was observed.

Example 18

The experiment of Example 17 was repeated, except that clusterintranscript was quantified using RT-PCR. The results are summarized inFIG. 17. Substantial reduction in the amount of clusterin transcript wasobserved.

Example 19

MCF-7 cells were transfected with various doses (5 and 50 nM) of CLU-5siRNA or scrambled control. Three days after treatment, proteins wereextracted and analyzed by Western blotting for clusterin levels (MW=40and 60 kDa). FIG. 18 shows the reduction in the amount of clusterinprotein in cells treated with the siRNA relative to the scrambledcontrol.

Example 20

Clusterin over expressing LNCaP)/T1 cells were transfected (1 pulse)with 10 nM CLU-5 siRNA or scrambled control. Three days after treatment,the proteins were extracted and analyzed by Western blotting forclusterin. No clusterin was detected in the cells treated with thesiRNA.

Example 21

3 species of siRNA targeting IGFBP-2 and -5 were formed asdouble-stranded RNA from Seq. ID NOs. 39 and 40, 41 and 42, and 43 and44 and labeled as BS-1-BS-3, respectively. A549 cells were transfectedwith various doses (10, 50 and 100 nM) of the 3 species of siRNA orscrambled control. A vehicle only control and an untreated control werealso evaluated. Total RNA was extracted and analyzed by RT-PCR forIGFBP-2 transcript. As shown in FIG. 19, reduction in the amount ofIGFBP-2 transcript was observed with all three species of siRNA.

Example 22

PC3 cells were transfected with various doses (10, 50 and 100 nM) of the3 species of bispecies siRNA (BS-1, BS-2 and BS-3) or scrambled control.A vehicle only control was also evaluated. Total. RNA was extracted andanalyzed by RT-PCR for IGFBP-5 transcript. As shown in FIGS. 20 and 21,reduction in the amount of IGFBP-5 transcript was observed with allthree species of siRNA.

Example 23

Primary human bone fiborblasts were transfected with 50 nM of the 3species of bispecific siRNA (BS-1, BS-2 and BS-3) or scrambled control.A vehicle only and an untreated control were was also evaluated. TotalRNA was extracted and analyzed by RT-PCR for IGFBP-5 transcript. Asshown in FIG. 22, reduction in the amount of IGFBP-5 transcript wasobserved with all three species of siRNA.

Example 24

C42 cells (a sub-line of LNCaP prostate cancer cells) were treated withBS-1, BS-2, and BS-3, and growth inhibition was assessed using a crystalviolet assay. The results are shown in FIG. 23.

Example 25

A549 lung cancer cells were treated with BS-1, BS-2, and BS-3, andgrowth inhibition was assessed using a crystal violet assay. The resultsare shown in FIG. 24.

1. An RNA molecule having a sequence effective to mediate degradation orblock translation of mRNA that is the transcriptional product of atarget gene, wherein the target gene encodes clusterin, and the RNAmolecule consists of sequence of bases as defined by Seq. ID No.
 6. 2. Apharmaceutical composition comprising an RNA molecule having a length ofless than 49 bases and having a sequence effective to mediatedegradation or block translation of mRNA that is the transcriptionalproduct of a target gene, wherein the target gene encodes clusterin, andthe RNA molecule consists of a sequence of bases as defined by Seq. IDNo. 6, together with a pharmaceutically acceptable carrier.
 3. Thepharmaceutical composition of claim 2, wherein the pharmaceuticallyacceptable carrier is a sterile injectable solution.
 4. An RNA moleculehaving a sequence effective to mediate degradation or block translationof mRNA that is the transcriptional product of a target gene, whereinthe target gene encodes clusterin, wherein the RNA molecule is doublestranded, and one of the strands consists of Seq ID No. 6.